CN102397301B - 使用酶的银耳提取物制备方法及含有它的记忆增进用组合物 - Google Patents
使用酶的银耳提取物制备方法及含有它的记忆增进用组合物 Download PDFInfo
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Abstract
本发明涉及使用酶的银耳提取物的制备方法及包含它的记忆增进用组合物,具体地说,涉及通过酶处理使制备收率得到提高的银耳提取物的制备方法及包含它的神经细胞恢复、神经细胞损伤抑制及记忆增进用食品组合物及药学组合物。
Description
技术领域
本发明涉及使用酶的银耳提取物制备方法及含有它的记忆增进用组合物,具体地说,涉及通过酶处理使收率得到提高的银耳提取物的制备方法及包含它的记忆增进用食品组合物及药学组合物。
背景技术
神经生长因子(Nerve growth factor,NGF)在中枢神经系统中起到保护神经细胞免受损伤的作用(J.Neurosci.Aug;6(8);2155-62;1986),对维持成熟的神经元起重要作用(Proc.Natl.Acad.Sci.USA vol.46;384-391;1960)。因此可以说,为使神经细胞在正常状态下生存、生长、分化,必须需要如神经生长因子的多种生长因子,上述神经生长因子能够预防和治疗痴呆,无现有治疗剂所具有的副作用,预期能够有效地发挥作用。然而上述神经生长因子由于分子量大,不能通过脑内的血脑屏障(Blood brain barrier,BBB),在实际临床中难以取得预期的治疗效果。因此实际上需要诱导内源性神经生长因子的合成,或者开发出发挥神经生长因子类似作用的物质。实际上,由天然物中探寻上述物质的研究正在进行中,并且本发明者也以此为线索报道了银耳提取物中的神经生长活性(kim et al.;Biol Pharm Bull.;30(4);708-714;2007)。
银耳(Tremella fuciformis)属于所谓的胶质菇(jelly mushroom),形成凝胶形态的子实体(fruit body)。银耳(Tremel lales)目(order)及银耳(Tremel laceae)科(family)所属的银耳(Tremella fuciformis)可用作食用菇。主要在夏季滋生于阔叶树的腐木。在包含韩国、日本等在内的全世界范围广泛分布。此外,银耳的子实体作为常用食品,在中国是作为强壮剂应用于临床的传统药物(Li,S.S.Chinese medicinal herbs;Georgetown Press:San Francisc)。其子实体成袋状,放大并相互融合形成带有水波状皱纹的块。呈浅黄色或黄色,偶见橙黄色,表面为弯曲的水波状但却平滑,在其整个表面产生子实体。尺寸通常为直径6cm、高3-4mm,但湿气较多时也可以生长得更大些。
本发明者在先前的报道中发现了银耳提取物具有神经细胞恢复及损伤抑制作用,完成了含有银耳提取物的记忆增进用组合物的相关发明。然而在依照现有报道中的银耳提取物的制备方法的情况下,存在着生产收率不高、难以应用于产品的困难。因此与现有的提取物进行比较,实际上需要研究出具有同等的或得到提高的记忆增进作用的同时,收率得到显著提高的银耳提取物的制备方法。
发明内容
要解决的技术问题
为此,本发明者在可提高银耳提取物制备收率的方法的研究过程中,在酸性条件下热水加压提取后进行酶处理时,发现了银耳提取物的收率显著提高。因此,本发明中在酸性条件下热水加压提取后,增加了酶处理工程,完成了收率得到提高的银耳提取物的制备方法。
因此,本发明的目的在于,提供一种由(a)在酸性条件下对银耳进行热水加压提取的步骤;及(b)对所述的热水提取物进行酶处理的步骤组成的银耳提取物的制备方法。
本发明的另一目的在于,提供一种依照所述的制备方法制备出的银耳提取物。
本发明的又一目的在于,提供一种含有所述的银耳提取物的记忆增进用食品组合物。
本发明的又一目的在于,提供一种含有所述的银耳提取物的记忆力障碍疾病预防及治疗用药学组合物。
技术方案
为实现上述本发明的目的,本发明提供了一种由(a)在酸性条件下对银耳进行加压热水提取的步骤;及(b)对所述的热水提取物进行酶处理的步骤组成的银耳提取物的制备方法。
本发明的另一目的在于,提供一种依照所述的制备方法制备出的银耳提取物。
本发明的又一目的在于,提供一种含有所述的银耳提取物的记忆增进用食品组合物。
本发明的又一目的在于,提供一种含有所述的银耳提取物的记忆力障碍疾病预防及治疗用药学组合物。
以下对本发明进行详细说明。
本发明涉及由(a)在酸性条件下对银耳进行热水加压提取的步骤;及(b)对所述的热水提取物进行酶处理的步骤组成的,与传统的提取方法相比收率显著提高的银耳提取物的制备方法。所述的各步骤具体说明如下。
步骤(a)是在酸性条件下热水加压提取银耳的步骤。
银耳(Tremella fuciformis)属于银耳科,夏季和秋季多生于阔叶树腐木。成白色,如明胶的半透明状,含有蛋白质、脂肪、碳水化合物、粗纤维、无机盐类等,已知具有恢复疲劳、免疫增进、降血糖及抗肿瘤作用,对便秘、腹泻、胃炎有效,此外还具有产后调理作用。
本发明中银耳具有增进记忆及神经细胞恢复作用。
本发明的银耳提取物的特征在于,在热水提取时,在酸性条件下进行提取。
本步骤中,为了使银耳更加易于提取而使用经过微粉的银耳。本发明中银耳可使用干品或直接使用鲜品。为使所述的银耳更加易于提取,可使用粉末或粉碎后使用,优选经过微粉的银耳。尽管粉碎时不受限制,但以使用锤式粉碎机(Hammer mill)为佳,对粒度大小不设限制,但以75至85目(mesh)为佳。本发明实施例中,在经过微粉的银耳中加入相当于所述的银耳的质量的40至60倍的水作为提取溶剂,调节溶剂的pH至酸性。这是为了在制备银耳提取物时,降低银耳中溶出的凝胶成分引起的粘性。粘性低的提取物与粘性高的提取物相比,具有溶解性及产品适用性得到提高,易于加工的优点。
尽管所述的酸性条件不受限制,但以pH 2.5至pH 3.5为佳,pH 2.7至pH 3.2更佳。尽管为调节pH可添加到所述的溶剂中的酸不受限制,但可以是枸橼酸、冰醋酸、苹果酸、乳酸、酒石酸、琥珀酸及富马酸等。
尽管所述的热水提取中的提取温度不受限制,但以110℃至130℃为宜,120℃至125℃更佳。此外,提取时间以15至25分钟为佳,18至22分钟更佳。
步骤(b)是所述的热水提取物的酶处理步骤。
本发明的特征在于,利用酶处理银耳的热水提取物,使提取物的收率提高。酶处理时,酶作用于组成银耳子实体的多糖体,切断它们之间的结合,从而降低银耳提取物制备时产生的特有的粘性,减少后续制备工程中可能产生的损失,具有最终提高生产收率的作用。
尽管本发明中可使用的酶不受限制,但可以是纤维素酶(cellulase)、果胶酶(pectinase)、木素降解酶(ligniase)、葡聚糖酶(glucanase)及半纤维素酶(hemicellulase),以使用纤维素酶为佳。纤维素酶主要是由霉菌、细菌及原生动物产生的酶,起分解纤维素的作用。本发明中的纤维素酶切断组成银耳子实体的多糖体之间的结合,降低提取物的粘性,具有提高生产收率的作用。
本步骤中,所述的酶处理以在酶的最佳活性条件下处理为佳。酶的最佳活性条件是酶的作用表现出最大活性的条件,是指温度、pH及压力等环境。本发明中,在pH 2.5至pH 6.0及40℃至60℃下进行所述的酶处理。此外,本发明中酶的最佳活性条件不限于特定范围,可根据处理酶设定该酶的最佳条件。即,作为本发明中可使用的酶的最佳活性条件,果胶酶在pH 2.5至pH 6.0、45℃至55℃下,木素降解酶在pH 2.5至pH 6.0、45℃至55℃下,葡聚糖酶在pH 4.5至pH 6.0、50℃至60℃下,半纤维素酶在pH 4.5至pH 5.0、50℃至60℃下具有最佳活性。特别是在本发明一实施例中,使用纤维素酶作为酶,在纤维素酶处理前,将pH设置在pH 4.5至pH 5.0,将温度设置在50℃至60℃。
作为用于调节所述的pH的物质,尽管不限于此,但可以选择氧化镁、碳酸氢钠、碳酸镁、沉降碳酸钙、合成水滑石、硅酸铝镁、偏硅酸铝镁、硅酸镁、合成硅酸铝、无水磷酸氢钙、磷酸氢钙、干燥氢氧化铝凝胶、氢氧化镁、氢氧化铝镁、氢氧化铝凝胶、氢氧化铝-碳酸氢钠共浸生成物、氢氧化铝-碳酸镁混合物干燥凝胶、氢氧化铝-碳酸镁-碳酸钙共浸生成物、金乌鲗骨、石决明及氨基乙酸中的一者使用。
本发明一实施例中为进行pH调节使用碳酸氢钠。
本步骤中尽管酶处理不限于此,但以处理10小时至15小时,优选12小时至14小时为佳。
此外,本发明可以包含使所述的步骤(b)中处理的酶失活的步骤。本发明中为提高收率进行的酶处理,如上所述仅进行一定时间,并在此后使酶失活。这是为了消除可能因酶引起的副作用的过程,酶因热发生变性而失活,因此以加热使其失活为佳。本发明一实施例中,为使纤维素酶失活,在100℃下加热10分钟。
本发明中,在制备所述的银耳提取物之后,还可以包含去除不溶性的银耳残留物的过程。在去除银耳残留物时经过使用凝聚剂使残留物沉降的过程,“凝聚剂”是指用于使悬浮在液体中的几个固体颗粒聚集形成略大的块而添加在液体中的药品。凝聚后液体中的颗粒通常其沉降速度加快,因此其用于促进漂浮物的沉降。
可用作所述的凝聚剂的物质不受限制,可以使用聚合氯化铝、硫酸铝、藻酸钠、聚合硫基硅酸铝、聚合羟基氯化硅酸铝、硫酸亚铁、氯化亚铁(液体)、聚胺、聚合羟基氯化硫酸铝、硅藻土或沸石,以使用硅藻土或沸石为佳。
本发明一实施例中,使用硅藻土使银耳的残留物沉降,使过滤更加容易。此外,本发明中添加凝聚物质使银耳的残留物沉降后,还包括了利用压滤机将其过滤的过程。
此外,本发明中为了使依照所述的制备方法制备的银耳提取物更加易于使用,还可以包含将其浓缩的过程。浓缩时,尽管不限于此,可以使用连续式浓缩器。
此外,本发明中还涉及依照所述的制备方法制备的银耳提取物。本发明中的银耳提取物可以直接使用,也可以根据需要干燥使用。所述的干燥时,尽管不限于此,但可以使用冷冻干燥法或热风干燥法,并且在热风干燥时,可添加业界公知的赋形剂。
此外,本发明中还涉及包含所述的银耳提取物的记忆增进用食品组合物。
“记忆增进”是指促进老化引起的阿尔茨海默病或其它内因、外因因素引起的受损神经细胞的活性,提高记忆力。本发明中的记忆增进是指,可以通过抑制神经细胞的损伤及恢复受损神经细胞实现的,神经生长因子的作用。已知本发明的银耳提取物,是诱导神经生长因子的合成,或起与其相似作用的,带有神经生长活性的物质。
所述的本发明的食品组合物,包括了功能性食品(functional food)、营养补充剂(nutritional supplement)、健康食品(health food)及食品添加剂(food additives)等所有形态。
所述的有形的食品组合物,可通过业界公知的普通方法制成多种形态。举例来说,作为健康食品,尽管不限于此,可将银耳提取物液体化、颗粒化、胶囊化及粉末化后摄取,制成茶、果汁及饮料形态饮用。此外,可将银耳提取物与已知具有神经细胞恢复、神经细胞损伤抑制及记忆增进作用的公知的活性成分一同混合,制成组合物形态。此外,作为功能性食品,尽管不限于此,可添加银耳提取物制造饮料(含酒精饮料)、水果及其加工食品(如:水果罐头、果酱等)、鱼类、肉类及其加工食品(如:火腿等)、面包类及面类(如:乌冬面、荞麦面、方便面、意大利面、通心粉等)、果汁、各种饮料、饼干、饴糖、乳制品(如:黄油、奶酪等)、使用植物油脂、人造奶油、植物性蛋白、冷冻食品、各种调味料(如:黄酱、酱油、沙司等)等。此外,为了以食品添加剂的形态使用银耳提取物,可制成粉末或浓缩液形态使用。
本发明的食品组合物中,银耳提取物的优选的含量不限于此,但添加量通常在最终制成食品中占到0.1至5重量%。本发明提取物的含量以健康及卫生为目的,或者以调节健康为目的,在长时间摄取的情况下,可以在所述的范围以下,有效成分在安全性方面不存在任何问题,因此确定也可以使用所述的范围以上的量。此外,含有本发明的银耳提取物作为有效成分的食品组合物,已知尤其具有神经细胞恢复、神经细胞损伤抑制及增进记忆的作用,能够与活性成分一同混合,制成健康食品形态。
此外,本发明中涉及包含所述的银耳提取物的记忆力障碍疾病预防及治疗用药学组合物。
本发明中的记忆力障碍疾病,是指表现出记忆力低下或减退症状的疾病。例如,所述的疾病可以包括阿尔茨海默病(Alzheimer’s disease)、痴呆(dementia)、健忘症(amnesia)、轻度认知障碍、亨廷顿氏病及帕金森病。
本发明中的药学组合物,可以单独含有本发明的组合物,或者还可以另外含有一种以上的药学上容许的载体、赋形剂或稀释剂。上述的“药学上容许”是指生理学容许,并且在给予人体时通常不引起过敏反应或与其类似的反应的组合物。
本发明中的记忆增进用药学组合物能够以任意方法给予哺乳动物。例如,可通过经口或非经口方式给药。非经口给药方法尽管不限于此,但可以是经皮给药、皮下注射、静脉注射、肌肉注射或腹腔注射。
本发明药学组合物如上所述,可根据给药途径制剂化,制成经口给药用或非经口给药用制剂。
在经口给药制剂的情况下,本发明的组合物可利用业界公知的方法剂型化,制成粉末、颗粒、片剂、丸剂、糖衣片剂、胶囊剂、液剂、凝胶剂、糖浆剂、糊剂、悬浊液等。例如,口服制剂可将活性成分与固体赋形剂混合,之后将其粉碎,添加适宜的辅料,之后加工成颗粒混合物,从而得到片剂或糖衣片剂。适宜的赋形剂例如可以包括:包含乳糖、葡萄糖、蔗糖、山梨醇、甘露醇、木糖醇、赤藓糖醇及麦芽糖醇等的糖类,包含玉米淀粉、小麦淀粉、大米淀粉及土豆淀粉等的淀粉类,包含纤维素、甲基纤维素、羧甲基纤维素钠及羟丙甲纤维素等的纤维素类,如明胶、聚乙烯吡咯烷酮等的填充剂。此外,还可以根据情况添加交联聚乙烯吡咯烷酮,琼脂、藻酸或藻酸钠等崩解剂。另外,本发明药学组合物中,还可以额外包含抗凝剂、润滑剂、湿润剂、香料、乳化剂及防腐剂等。
在非经口给药用制剂的情况下,可利用业界公知的方法剂型化,制成注射剂、乳剂,洗剂,外用软膏剂、油剂、保湿剂、凝胶剂、气溶胶及鼻腔吸入剂的形态。上述剂型记录在所有制药化学中普遍公知的处方文献(Remington’s Pharmaceutical Science,15thEdition,1975.Mack Publishing Company,Easton,Pennsylvania 18042,Chapter 87:Blaug,Seymour)中。
本发明的组合物的总有效量可以单一剂量(single dose)给予患者,也可以通过以多次给药(multiple dose)方式长时间给药的分步治疗法(fractionated treatment protocol)给予患者。本发明药学组合物根据记忆增进程度的需要,可以改变有效成分的含量。优选地,本发明的组合物的体积容量,以每日患者体重每kg约0.01μg至1,000mg为佳,以0.1μg至100mg为最佳。然而所述的组合物的浓度,不仅要考虑到药学组合物的给药途径和治疗次数,还要考虑到患者的年龄、体重、健康状态、性别、疾病重症度、饮食及排泄率等多种因素,以确定患者的有效给药量,因此在考虑上述几点时,具有该领域普通知识的技术人员,可根据所述的组合物作为记忆增进剂的特定用途,决定合适的有效给药量。本发明中的药学组合物并不特别受限于表现本发明的作用的剂型、给药途径及给药方法。
因此,本发明中的银耳提取物可用于使记忆力得到增强,或者记忆力障碍疾病的预防或治疗中。即,含有本发明中的银耳提取物的组合物,可用于增强正常人的记忆力或者预防记忆力障碍疾病,也可以用于具有记忆力障碍疾病的患者的治疗。所述的银耳提取物是对人体几乎不表现出毒性,非常安全的物质,因此可制成药学组合物或食品组合物等形态。
有益效果
如上所述,本发明中,涉及使用酶的银耳提取物的制备方法,及包含它的记忆增进用组合物,具体地说,涉及通过酶处理使收率得到提高的银耳提取物的制备方法,及包含它的神经细胞恢复、抑制神经细胞损伤及记忆增进用食品组合物及药学组合物。
附图说明
图1所示为本发明的银耳提取物的制备过程的一实施例。
图2所示为以PC12h细胞株的神经轴突的生长程度示意的本发明的银耳提取物的神经生长活性作用,图2(A)所示为对照组,图2(B)所示为神经生长因子处理组,图2(C)所示为本发明的银耳提取物处理组,图2(D)所示为现有方法的银耳提取物处理组。
具体实施方式
以下参照实施例对本发明进行详细说明。
但,以下实施例仅用于揭示本发明,本发明的内容并不限于以下实施例。
<实施例1>
通过纤维素酶的处理制备银耳提取物
将银耳40kg细致粉碎,添加相当于银耳质量50倍的水。在其中添加4kg的枸橼酸,使pH达到3.0,之后升高温度,在121℃下进行20分钟的提取。在其中加入碳酸钾2kg,使pH达到4.5至5.0。降低提取液的温度至55℃,加入纤维素酶4kg,搅拌12hr以上,同时进行酶反应。之后为使酶失活,以100℃再加热10分钟。为了去除提取物内的不溶性物质,加入硅藻土,以压滤机(韩国过滤器(株))过滤,之后在连续式浓缩器(韩特(株))中浓缩至8糖度。确认将其冷冻干燥得到的银耳提取物的含量。所述的过程如图1所示。
另外,为了比较所述的提取方法和收率,还通过传统的热水提取方法制备出银耳提取物,确认了提取物的制备收率。将40kg的银耳细致粉碎,添加相当于银耳质量50倍的水,在90至100℃下进行热水提取。之后过滤去除银耳残留物,之后将其浓缩。之后确认了通过冷冻干燥得到的银耳提取物的含量。
其结果如表1所示,使用传统的热水提取方法的银耳提取物的收率仅为约5%,使用本发明的提取方法的收率约为81.5%,可知收率提高了15倍以上。
【表1】
提取方法 | 生产收率(以固形份为准) |
酶处理热水提取方法(本发明) | 81.5% |
热水提取方法 | 5% |
<实施例2>
神经细胞分化促进作用(Neuritogenic Effect)测定
将所述的实施例1中制备的本发明银耳提取物(银耳提取物1)和依照传统提取方法制备的银耳提取物(银耳提取物2)粗制成1μg/mL的浓度,对未分化的PC12h细胞(subclone ofPC12 cells isolated by Dr.Hatanaka,Rat)进行处理,在37℃、5%CO2培养基中培养48小时。培养后利用0.1%戊二醛(glutaraldehyde)溶液固定细胞,利用影响分析仪(Imageanalyzer)(Leica,Germany)测定延伸的神经细胞突起的长度。各实验组任意选定五部分,依次测定该部位中最长的10个神经细胞突起。测得的神经突起的长度通过ANOVA test和T-test(Yamazaki et al.;Biol.Pharm.Bull.;vol.19;page 791-795;1996)验证其有义性。
其结果如表2所示,作为对照组使用的PBS处理组的平均神经突起长度为95.38±5.08μm,本发明的银耳提取物(银耳提取物1)处理组中的平均神经突起的长度为107±3.63μm,与对照组PBS处理组进行比较时,表现出有意义的高神经生长活性作用,确认了类似于以传统方法制备的银耳提取物(银耳提取物2)的水平。由此可以确认,通过本发明能够以高收率制备神经生长活性作用优秀的银耳提取物。所述的实验结果在表2中一并示意。
【表2】
1)银耳提取物1:本发明银耳提取物
2)银耳提取物2:现有报道的银耳提取物
*P<0.01,Significantly different from the control group.(ANOVA,Scheffe’s test,n=300)
1)神经细胞突起的平均长度±S.E.(μm)
<实施例3>
银耳提取物的粘度测定的粘度测定
测定了本发明的银耳提取物和以传统方法制备的银耳提取物的粘度特性变化。分别以50mg/mL的浓度溶解两种提取物,之后利用旋转型粘度计在25℃、100rpm下测定其粘度。
其结果如表3所示,传统的基于热水提取的银耳提取物的粘度为19,960,相比之下,通过本发明得到的银耳提取物的粘度不超过120,提示粘度与传统的银耳提取物相比得到减少。
【表3】
提取方法 | 粘度(cps) |
酶处理热水提取方法(本发明) | 120 |
热水提取方法(传统发明) | 19,960 |
应用前景
本发明涉及使用酶的银耳提取物的制备方法及包含它的记忆增进用组合物。本发明中,提供了通过酶处理使收率得到提高的银耳提取物的制备方法,及通过所述的方法制备的银耳提取物,它可用作神经细胞恢复、抑制神经细胞损伤及记忆增进用食品组合物及药学组合物。
Claims (2)
1.一种提高神经细胞分化促进作用的银耳提取物的制备方法,其特征在于包括:
(a)对银耳添加枸橼酸,在pH2.5至pH3.5的酸性条件下及在110℃至130℃的温度下进行加压热水提取的步骤;及
(b)对所述(a)步骤的热水提取物加入碳酸钾,在pH2.5至pH6.0及40℃至60℃下进行酶处理的步骤;所述的酶选自由纤维素酶、果胶酶、木素降解酶、葡聚糖酶及半纤维素酶组成的群。
2.根据权利要求1所述的制备方法制备的银耳提取物。
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